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Abstract

Structured illumination microscopy provides a simple and cheap mean to obtain optical sections of a sample. It can be implemented easily on a regular fluorescent microscope and is a scanning free alternative to confocal microscopy. We have analyzed theoretically the performances of the technique in terms of sectioning strength, resolution enhancement along the optical axis, and signal to background as a function of the objective used and the grid’s characteristics (pitch and contrast). We show that under optimized conditions, the axial resolution can be improved by a factor of 1.5 in comparison with an epifluorescence microscope, and that optical cuts with a thickness of less than 400nm can be obtained with a 1.4 numerical aperture objective. We modified the original grid in-step modulation pattern and used a sinusoidal modulation for the grid displacement. Optical sections are computed by combining four images acquired during one modulation period. This algorithm is very stable even for modulations at high frequencies. The speed of the acquisition is thus only limited by the performance of the detector and the signal/background ratio of the sample. Finally, we compared our technique to commercial setups: a confocal microscope, a Spinning Disk Microscope and a Zeiss Apotome.

Figures (5)

Fig. 1. FWHM of the axial response in optical units as a function of the normalized spatial frequency of the grid. This shows that the narrowest axial response is achieved for a normalized spatial frequency equal to 1

Fig. 2. PSF and OTF for an epifluorescence microscope and for structured illumination: the OTF of structured illumination microscope (bottom right) is enlarged compared to the OTF in the epifluorescence case (top right). This is consistent with the PSF of the structured illumination case that is narrower along the optical axis than that of the epifluorescence case

Tables (1)

Table 1. Axial response in microns and corresponding υ′g: this table summarize the achievable thicknesses evaluated as the FWHM of the axial response (given in microns) for different grid pitches (given in line pair per millimeter) for different objectives and grids. We also indicate the corresponding normalized spatial frequency of the grid in the plane of the specimen υ′g which was calculated accordingly to our experimental setup. The wavelength λ was set to 0.6µm.

Metrics

Table 1.

Axial response in microns and corresponding υ′g: this table summarize the achievable thicknesses evaluated as the FWHM of the axial response (given in microns) for different grid pitches (given in line pair per millimeter) for different objectives and grids. We also indicate the corresponding normalized spatial frequency of the grid in the plane of the specimen υ′g which was calculated accordingly to our experimental setup. The wavelength λ was set to 0.6µm.